Dissecting the Conformational Stability of a Glycan Hairpin

Systematic structural studies of model oligopeptides revealed important aspects of protein folding and offered design principles to access non-natural materials. In the same way, the rules that regulate glycan folding could be established by studying synthetic oligosaccharide models. However, their analysis is often limited due to the synthetic and analytical complexity. By utilizing a glycan capable of spontaneously folding into a hairpin conformation as a model system, we investigated the factors that contribute to its conformational stability in aqueous solution. The modular design of the hairpin model featured a trisaccharide turn unit and two β-1,4-oligoglucoside stacking strands that allowed for systematic chemical modifications of the glycan sequence, including the introduction of NMR labels and staples. Nuclear magnetic resonance assisted by molecular dynamics simulations revealed that stereoelectronic effects and multiple glycan–glycan interactions are the major determinants of folding stabilization. Chemical modifications in the glycan primary sequence (e.g., strand elongation) can be employed to fine-tune the rigidity of structural motifs distant from the modification sites. These results could inspire the design of other glycan architectures, with implications in glycobiology and material sciences.


■ INTRODUCTION
Synthetic oligomers capable of folding into defined conformations (i.e., foldamers) are valuable models to dissect the intricate rules that regulate biopolymer folding. 1,2These discrete molecules offer well-defined chemical structures and modularity of synthesis and allow for systematic chemical modifications to establish correlations between the primary sequence and secondary structures. 3For example, synthetic peptide hairpins improved our understanding of the factors influencing folding, 2,4 stability, 5 and aggregation 6 of beta-sheet containing proteins (e.g., amyloids 7 ).Parallel 8 or antiparallel 4 hairpins (Figure 1) as well as more complex three-strand betasheets systems 9 were designed, and their conformational stability was dissected through systematic chemical modifications.These systems revealed the origin of the beta-sheet stability as a complex combination of interstrand hydrogen bonds, 10 van der Waals and hydrophobic interactions, 11 and conformational proclivity of the backbone (i.e., stereoelectronic effects and entropic factors). 12These works also offered design principles to construct synthetic analogues capable of spanning non-natural geometries. 13,14n contrast to peptides, the rules that regulate glycan folding are much more obscure, because these compounds exist in a dynamic ensemble of conformers separated by low energy barriers. 15In addition, the lack of synthetic standards and the complexity of glycan analysis have hampered systematic structural studies. 16,17Nevertheless, insights into the fundamentals of glycan folding are extremely valuable to understand glycan properties 18 and design synthetic analogues capable of performing a particular function. 19cently, we have designed and synthesized a glycan sequence, capable of spontaneously folding into a hairpin conformation (Figure 1). 20The design featured a rigid trisaccharide turn motif substituted with two β-1,4-oligoglucoside stacking strands.NMR analysis unequivocally confirmed its folded conformation in an aqueous solution.The modular design of the glycan hairpin offers an ideal model system to systematically investigate the determinants of its conformational stability and identify design principles to access new folded architectures.
Herein, we dissect the underlying forces that stabilize glycan folding by using a glycan hairpin model.We prepared a collection of synthetic hairpin analogues to systematically analyze (i) the chemical structure of the turn unit, (ii) the interstrand interactions between the β-1,4-glucan strands, and (iii) the strands' length influence on conformational stability (Figure 1).All hairpin analogues were synthesized with automated glycan assembly (AGA) 21,22 ; the collection featured 13 C-labeled analogues 16,23 as well as a stapled glycan hairpin to serve as a "fully folded" reference structure. 24Structural analysis was performed with nuclear magnetic resonance (NMR) spectroscopy, assisted by molecular dynamics (MD) simulations.We reveal that glycan−glycan interactions can be exploited to rigidify glycan sequences and that simple chemical modifications can dramatically influence the stability of glycan motifs remote from the modification site.These rules can inspire the creation of novel glycan foldamers as well as glycomimetics with increased conformational stability. 19

■ RESULTS AND DISCUSSION
A hairpin can be conceptualized as a turn unit substituted with two stacking strands.Herein, we discuss how systematic modifications of these structural motives affect hairpin conformational stability to pinpoint the major determinants of folding stabilization.
All hairpins were synthesized by AGA on solid support (see Supporting Information Section 3) in an overnight run using previously reported conditions. 20,21Post-AGA manipulations included solid-phase methanolysis, photocleavage from the solid support, and hydrogenolysis (see Supporting Information Section 3).A single purification step afforded the target hairpin analogues in overall yields of 4−55%.Atomistic MD simulations were carried out to probe the tendency of the designed glycans to adopt a hairpin conformation.All the modeled structures were simulated for 500 ns unless otherwise specified, employing a modified version of the GLYCAM06 26 carbohydrate force field.The systems were solvated with the TIP5P 27 water model to avoid excessive interactions between the monomers. 28A systematic terminology to standardize the name and representation of the glycan hairpins discussed in this paper can be found in the SI (Figures S2 and S3).Labeling of protons in a monosaccharide is done as follows: e.g., the proton attached to C-1 of Rha is named "Rha-1".
The Effect of the Turn.The turn unit of 9mer-I is inspired by the Le x trisaccharide 29 and contains a reducing D-GlcNAc branching unit substituted with a β-D-Glc unit at C-4 and an α-L-Rha unit at C-3 (Figure 2).A nonconventional CH•••O hydrogen bond between the H-5 of L-Rha (Rha-5) and the O-5 pyranose oxygen of Glc-B stabilizes the turn, forming a 10-membered ring. 30This weak interaction is complemented by stereoelectronics of the glycosidic bonds (i.e., exoanomeric effect), the van der Waals and hydrophobic interactions between the methyl group of Rha and the hydrophobic face of Glc, and a steric contribution connected to the amide moiety of GlcNAc. 30,31This turn proved suitable to hold two cellulose branches in an ideal parallel orientation resulting in a folded hairpin conformation. 20e designed and synthesized new hairpin analogues carrying the same cellulose strands to investigate the effect of specific chemical changes at the turn unit on the overall hairpin conformational stability (Figure 2 and see Supporting Information Section 3).9mer-I-Linker carries an aminopentanol linker 32 at the reducing end preventing anomerization.9mer-II is a control structure missing the Rha unit (i.e., lacking the unconventional H-bond).9mer-III is based on a reducing D-Glc branching unit (instead of GlcNAc) lacking the stabilizing acetyl amido group.In 9mer-IV, the position of the branches (L-Rha and D-Glc) is inverted to promote a different orientation of the two stacking strands.The inversion of L-Rha and D-Glc should preserve the close conformation of the turn, as shown for the trisaccharide regio-isomers Le x and Le a . 31ll compounds were modeled computationally by MD simulations.For each structure, the average distance between residues on the opposite strands was measured, plotted, and compared with the values obtained for 9mer-II, which adopts an open conformation. 20MD simulations indicated no significant differences for the four hairpins containing a Rha unit (Figure 2a and Figures S7−S10), all adopting the closed conformation for most of the simulation time.
The chemical shift deviation (Δδ) of Rha-5 served as an experimental marker of the closed conformation. 30,33We monitored the chemical shift of Rha-5 for all 9mer hairpins and compared it to a control trisaccharide lacking the H-bond 3mer-V (δ = 4.06 ppm).All compounds preserved the nonconventional H-bond over a wide temperature range, as indicated by the downfield shift of Rha-5 (Δδ ∼ 0.4 ppm) (Figure 2b and Table S4).Moreover, the presence of a key inter-residue NOE Rha-6/Glc B-2 confirmed the conformational stability of all turn motives (Figures S44, S49, and S52).
Overall, the similar results obtained for 9mer-I and 9mer-III suggested the limited contribution of the amido group to hairpin stabilization.Similarly, a comparison between 9mer-I and 9mer-I-Linker demonstrated that anomerization is not a source of flexibility.The largest chemical shift deviation was detected for 9mer-IV.This observation indicated a higher conformational stability for the 9mer-IV analogue, in agreement with the reported conformational stability of Le a vs Le x . 31nalysis of the MD trajectories indicated a twisted conformation of the two strands with opposite directionality Autonomously folding antiparallel or parallel peptide hairpin model systems have been widely explored in terms of conformational stability, whereas less is known about the rules that regulate glycan folding.Herein, we explore the main determinants of the conformational stability of a model glycan hairpin.The following abbreviations are used for monosaccharides: Glc = glucose (blue circle), GlcNAc = N-acetyl glucosamine (blue square), and Rha = rhamnose (green triangle).The monosaccharide residues are represented following the Symbol Nomenclature for Glycans (SNFG). 25or 9mer-III and 9mer-IV (Figure S12).Further investigations are needed to confirm such helical propensity and its impact on hairpin stabilization.More drastic differences were detected when comparing shorter hairpins (5mer-I vs 5mer-III, Figure S33), indicating a major contribution of strand−strand interactions to the overall hairpin conformation (see Supporting Information Section 4.3.2 for a detailed comparison).Thus, we moved on to analyze the stabilizing effect of the strands.
The Effect of the Strands.To analyze the stabilizing effect of strand−strand interactions, experimental NMR data for 9mer-III and 6mer-III were compared.The latter is a truncated hairpin composed of a turn unit but lacking one of the strands.The Rha-5 chemical shift was used as an indicator of turn rigidity, showing a higher value for 9mer-III (δ = 4.45 ppm) in comparison to 6mer-III (δ = 4.37 ppm) (Table S4).The difference between the two compounds became even more evident at a higher temperature as the gap between Rha-5 chemical shifts increased (Figure 3a).In contrast, the behavior of 6mer-III was virtually identical to that of 3mer-III, the control compound lacking both strands.1D-selective TOCSY-NOESY experiments were performed to further corroborate this observation (Figure 3b).Selective irradiation of Rha-6 followed by TOCSY transfer of magnetization to Rha-5 and then NOESY from Rha-5 permitted us to measure the intraresidue Rha-5/Rha-3 (as reference) and the interresidue Rha-5/Glc B-2 NOEs for 6mer-III and 9mer-III.
The NOE data confirmed the shorter Rha-5/Glc B-2 distance in 9mer-III (2.51 Å) than that in 6mer-III (2.71 Å) (Figure 3b), demonstrating that strand−strand interactions rigidify the turn motif.Besides the turn, the close proximity between the two strands is encoded in the Glc C-1 chemical shift (Figure S72).Upfield Glc C-1 shifts (δ ≤ 4.40 ppm) are observed only when residue Glc D on the opposite strand was present, while for the 6mer-III, it was significantly downfield shifted (δ 4.45 ppm).
Ramachandran plots for a Glc−Glc glycosidic bond within the strands were compared for 9mer-III, 6mer-III, and a cellulose hexamer as control.While the linkage in 6mer-III showed a similar dihedral distribution with the cellulose hexamer, 9mer-III displayed a substantial shift toward negative Ψ values (Figure S13).This indicated that interactions between strands promoted the flattening of the structure, thus favoring the formation of the conventional H-bond between Glc residues. 34Overall, these experimental results confirmed the important contribution of strands' interactions in the stabilization of the hairpin conformation.
The Effect of Strand Length.Having proved the contribution of the strand−strand interactions on the overall conformational stability of the hairpin, we moved on to analyze the effect of the strands' length.A length-dependent stabilization has been demonstrated for specific peptide sequences folding into a hairpin conformation.In anti-parallel beta-sheet models, strands composed of five to seven residues provided optimal stabilization and further elongation did not lead to an increased stabilization but rather a loss of conformational rigidity. 35This behavior was not observed in parallel-β-sheet peptide models, which become steadily more stable as the strands are lengthened. 36o analyze the correlation between conformational stability and strand length, we prepared a collection of hairpins based on 3mer-III substituted with strands of increasing length (5mer-III to 17mer-III).This series was complemented with a stapled glycan hairpin (5mer-III-Closed) where the nonreducing ends were covalently connected with an aliphatic chain.This compound served as a "fully folded" reference structure as commonly done for the structural analysis of peptide hairpins. 24,37er-III-Closed was prepared by AGA following a ring closing metathesis (RCM) protocol. 38MD simulations of 5mer-III were analyzed to determine the most suitable hydroxyl groups for the introduction of the alkene moieties.
To constrain our hairpin in the closed conformation, we aimed to staple two hydroxyl groups pointing in the same orientation for most of the simulation time.Analysis of the dihedral angles O top,n − C top,n − C bottom,m − O bottom,m and the average distance of the corresponding oxygen atoms identified the hydroxyl groups at C top,4 and C bottom,4 as ideal positions for stapling (see Supporting Information Section 4.3.3 for detailed analysis).Thus, 5mer-III-Closed was prepared using the corresponding C-4 alkene-functionalized BBs (Figure S1 and see Supporting Information Section 3).
With this collection in hand, we set to identify whether the conformational stabilization increases linearly or whether there is an optimal length after which stabilization is lost.First, we focused on hairpins with shorter strands.The conformation of 5mer-III and 5mer-III-Closed in aqueous solution was compared using NMR spectroscopy (Figures S28 and S38).A downfield shift was observed for Rha-5 of 5mer-III-Closed (4.49ppm) as compared to 5mer-III (4.43 ppm) (Figure 4 and Table S4).The downfield shift for Rha-5 was linearly correlated with a high field 1 H NMR chemical shift for Glc C-1 (Figure S72).2D ROESY and TOCSY NMR experiments were performed to confirm the spatial proximity between key inter-residue protons of the turn and the strands.The analysis of ROESY 39 spectra showed inter-residue NOE signals (Glc B-2/Rha-6 and Glc C-1/Glc D-2) for both structures, albeit of different intensities (Figure 4b).Indeed, a closer interstrand distance was calculated for 5mer-III-Closed (2.63−2.74Å, Figure 4c,f) in comparison to 5mer-III (2.93−3.02Å, Figure 4a,f).Overall, these results indicated a certain degree of flexibility for hairpins with short strands.

Journal of the American Chemical Society
Next, we explored the effect of longer strands on the overall hairpin conformation.The chemical shift of Rha-5 (4.45 ppm for structures longer than 7mer) showed stabilization of the turn motif with strand elongation (Figure 4 and Table S4).Similarly, the comparison of inter-residue distances for all the simulated hairpin-III structures identified an increased conformational rigidity for the hairpin cores with longer strands (Figure 4d).While 5mer-III was in equilibrium between closed and a few open forms, longer analogues were closed for the majority of the simulation time (Figures S20− S23).To support the MD simulation results with solution-state NMR, we looked for the interresidue NOEs in short vs long hairpin structures.To simplify the NMR analysis, we introduced a 13 C-labeled residue 40 (highlighted in red color, Figure 4e) in the same position of the bottom strand in a short and in a long hairpin (7mer-III-F and 13mer-III-F).The use of a 13 C-labeled unit (Glc F) allowed us to identify the NMR signals for its contiguous Glc D residue and to calculate the interstrand distance between Glc C-1 and Glc D-2 (Figure 4f).A shorter distance was estimated for the long 13mer-III-F (2.88−2.98Å) than for the short 7mer-III-F (2.90−3.00Å).Moreover, the presence of 13 C-labels in residue F was instrumental to detecting a key interstrand NOE between Glc F-6 and Glc E-2.This NOE was detected only for the long 13mer-III-F (Figure S69).In contrast, the analogous NOE (between Glc F-6 and Glc E-2) could not be detected in the shorter 7mer-III analogue, likely due to the existence of internal motions of the E and F units.Given the dependence of the NOEs on the effective correlation times, these fast motions at the edges of 7mer-III result in smaller and undetectable NOEs.Taken together, these analyses indicated that the glycan hairpin becomes steadily more stable as the strands are lengthened.
Besides increasing conformational stability, long cellulose strands could engage in intermolecular interactions. 41DOSY analysis confirmed the aggregation tendency for longer hairpin analogues (Figure 5a).While the diffusion coefficient decreased linearly for short structures, a significant jump was detected for 13mer-III, suggesting the presence of certain aggregation.This result is in agreement with the observation of a precipitate forming during the synthesis, drastically decreasing the yield of the long analogues (17mer-III).Powder XRD analysis showed a crystalline packing for analogues longer than 9mer, with 13mer-III resembling the cellulose II crystal structure 42 and 15mer-III showing a mixture of two allomorphs (i.e., cellulose II and cellulose IV II ) 43 (Figure 5b).This tendency may be exploited in the future for the controlled formation of carbohydrate assemblies. 44onclusions.We analyzed the factors that influence the conformational stability of a glycan hairpin.The modularity of the hairpin design allowed us to systematically scrutinize the effect of chemical changes in the turn or in the strand length.We discovered that the conformational proclivity of the turn motif is fundamental to promoting folding but that some modifications (e.g., inversion of the strand orientation or functionalization of the reducing end) are tolerated and could be exploited to tune the hairpin geometry or for further functionalization.We identified the substantial contribution of strand−strand interactions to the overall hairpin conformational stability, which increased with the strand length.These results demonstrate that weak glycan−glycan interactions can be exploited to rigidify glycan sequences, normally considered to be highly flexible molecules.This finding is valuable for the design of rigid glycomimetics, capable of minimizing the entropic penalty of binding. 45Our systematic analysis also revealed the aggregation tendency and formation of crystalline materials for hairpins longer than 13mer.This feature could inspire the design of carbohydrate materials with tunable geometries, 44 as extensively explored for peptide hairpins. 13,46

Figure 1 .
Figure 1.Comparison between peptide and glycan hairpins:Autonomously folding antiparallel or parallel peptide hairpin model systems have been widely explored in terms of conformational stability, whereas less is known about the rules that regulate glycan folding.Herein, we explore the main determinants of the conformational stability of a model glycan hairpin.The following abbreviations are used for monosaccharides: Glc = glucose (blue circle), GlcNAc = N-acetyl glucosamine (blue square), and Rha = rhamnose (green triangle).The monosaccharide residues are represented following the Symbol Nomenclature for Glycans (SNFG).25

Figure 2 .
Figure 2. The effect of the turn: Representation of the structures of the 9mer hairpins with the different turn units analyzed in this work (for synthetic details, see the SI).(a) Average inter-residue distances calculated from the MD simulation trajectories (25,001 frames, 500 ns), showing an overall closed conformation for all 9mer apart from 9mer-II, a control structure lacking the Rha unit.Error bars represent the standard deviation.(b) Temperature-dependent 1 H NMR analysis of 9mer-I, 9mer-I-Linker, 9mer-III, and 9mer-IV.The chemical shifts of Rha-5 were extracted from selective 1D TOCSY (278, 298, 318 K, 700 MHz, d9 200 ms, D 2 O) showing that the nonconventional H-bond is preserved over a wide range of temperatures.

Figure 4 .
Figure 4.The effect of strand length: A collection of hairpin analogues with increasing strand length from 5mer-III to 17mer-III and a stapled glycan hairpin 5mer-III-Closed as a "fully folded" reference.(a) Superimposed 2D ROESY (blue) and 2D TOCSY (grey) NMR of 5mer-III showing the key interstrand NOEs with calculated distances (293 K, mixing times p15 300 ms and d9 160 ms, D 2 O, 700 MHz).(b) Experimental interstrand NOEs extracted form ROESY NMR experiments for 5mer-III and 5mer-III-Closed (red arrows).(c) Overimposed 2D ROESY and 2D TOCSY NMR of 5mer-III-Closed (293 K, mixing times d9 150 ms and p15 300 ms, D 2 O, 700 MHz).(d) Average inter-residue distances calculated by MD showing a trend of increased stabilization for hairpin core with increasing strands length.(e) Overimposition of two representative snapshots extracted from MD simulation showing that the cores (light blue) of the longer hairpin (13mer-III-F) are more rigid than those of the shorter hairpin (7mer-III).The highlighted (red) F residue indicates the 13 C-labeled unit.(f) Comparison of interstrand NOE (Glc C-1/Glc D-2) distance for 5mer-III, 5mer-III-Closed, 7mer-III-F, and 13mer-III-F.The calculated interstrand NOE distance for the 5mer-III-Closed hairpin is closer than that for 5mer-III, showing a certain degree of flexibility in shorter hairpin, while stability increases with strand elongation.